1. Field of the Invention
The invention provides nucleotide sequences from coryneform bacteria which code for the metR and metZ genes and a process for the fermentative preparation of amino acids, in particular L-methionine, by attenuation of the metR and/or metZ gene.
2. Description of the Related Art
L-Amino acids, in particular methionine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation process. Improvements to the process can relate to fermentation measures, such as stirring and supply of oxygen, or to the composition of the nutrient media, such as, the sugar concentration during the fermentation, or to the working up of the product by, for example, ion exchange chromatography, or to the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and which produce amino acids, such as e.g. L-methionine, are obtained in this manner.
Recombinant DNA techniques have also been employed for some years for improving of Corynebacterium strains which produce L-amino acids, by amplifying individual amino acid biosynthesis genes and investigating their effect on the amino acid production.
One object of the present invention is to provide new measures for improved fermentative preparation of amino acids, in particular L-methionine.
Where L-amino acids or amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine.
When L-methionine or methionine are mentioned in the following, the salts, such as methionine hydrochloride or methionine sulfate are also meant.
The invention provides isolated polynucleotides from coryneform bacteria, which comprise the polynucleotide sequences which code for the metR and/or metZ genes, chosen from the group consisting of
a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
b) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 3,
c) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID No. 2,
d) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID No. 3,
e) polynucleotide which is complementary to the polynucleotides of a), b) c) or d), and
f) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b), c), d) or e),
and the corresponding polypeptides according to a) or c) having the enzymatic activity of the transcription activator MetR and the polypeptides according to b) or d) having the enzymatic activity of O-succinylhomoserine sulfhydrylase (MetZ).
The invention also provides the above-mentioned polynucleotides, as DNAs which are capable of replication, comprising:
(i) the nucleotide sequence shown in SEQ ID No. 1, or
(ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequences complementary to sequences (i) or (ii), and optionally
(iv) sense mutations of neutral function in (i).
The invention also provides:
a DNA which is capable of replication and comprises the nucleotide sequence as shown in SEQ ID No. 1,
a polynucleotide that codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2 or SEQ ID No. 3,
a vector containing parts of the polynucleotide according to the invention, but at least 15 successive nucleotides of the sequence claimed
and coryneform bacteria in which the metR gene and/or the metZ gene is or are attenuated, in particular by deletion, insertion or base exchange.
The invention also provides polynucleotides which are obtained by screening a corresponding gene library of a coryneform bacterium, which comprises the complete gene or parts thereof by means of hybridization, with a probe which comprises the sequence of the polynucleotide according to the invention according to SEQ ID No. 1 or a fragment thereof, and isolation of the polynucleotide sequence mentioned.